Chondrogenic differentiation of human bone marrow-derived mesenchymal 1 stromal cells in a three-dimensional environment 2 3 Running title : Chondrogenic differentiation MSCs in 3 D 4 5

23 Cell therapy combined with biomaterial scaffolds is used to treat cartilage defects. We 24 hypothesized that chondrogenic differentiation bone marrow-derived mesenchymal stem 25 cells (BM-MSCs) in three-dimensional biomaterial scaffolds would initiate cartilaginous 26 matrix deposition and prepare the construct for cartilage regeneration in situ. The 27 chondrogenic capability of human BM-MSCs was first verified in a pellet culture. The 28 BM-MSCs were then either seeded onto a composite scaffold rhCo-PLA combining 29 polylactide and collagen type II (C2) or type III (C3), or commercial collagen type I/III 30 membrane (CG). The BM-MSCs were either cultured in a proliferation medium or 31 chondrogenic culture medium. Adult human chondrocytes (ACs) served as controls. After 32 3, 14, and 28 days, the constructs were analyzed with qPCR and confocal microscopy and 33 sulfated glycosaminoglycans (GAGs) were measured. The differentiated BM-MSCs 34 entered a hypertrophic state by day 14 of culture. The ACs showed dedifferentiation with 35 no expression of chondrogenic genes and low amount of GAG. The CG membrane 36 induced the highest expression levels of hypertrophic genes. The two different collagen 37 types in composite scaffolds yielded similar results. Regardless of the biomaterial 38 scaffold, culturing BM-MSCs in chondrogenic differentiation medium resulted in 39 chondrocyte hypertrophy. Thus, caution for cell fate is required when designing cell40 biomaterial constructs for cartilage regeneration. 41